1. Field of the Invention
The present invention relates to an isolated and purified DNA encoding a humanized bioluminescent green fluorescent protein (hPtFP) derived from the orange seapen Ptilosarcus gurneyi in which all the codons are the favored or most favored codons for mammalian expression systems. Truncation mutants of the humanized Ptilosarcus gurneyi fluorescent protein (hPtFP) of the present invention are functional as fluorescent reporter molecules in a biosensor system. The green fluorescent protein of the present invention is useful as an improved fusion partner in cellular proteins allowing direct observation of the behavior of the tagged protein.
2. Description of the Background Art
A major component of the new drug discovery paradigm is a continually growing family of fluorescent and luminescent reagents that are used to measure the temporal and spatial distribution, content, and activity of intracellular ions, metabolites, macromolecules and organelles. Classes of these reagents include labeling reagents that measure the distribution and amount of molecules in living or fixed cells, environmental indicators to report signal transduction events in time and space, and fluorescent protein biosensors to measure target molecular activities within living cells. A multiparameter approach that combines several reagents in a single cell is a powerful new tool for drug discovery.
Those skilled in this art will recognize a wide variety of fluorescent reporter molecules that can be used in the field of drug discovery. Particularly, herein are disclosed novel humanized fluorescent proteins. Similarly, fluorescent reagents specifically synthesized with particular chemical properties of binding or association have been used as fluorescent reporter molecules. (Barak et al., (1997), J. Biol. Chem. 272:27497–27500; Southwick et al., (1990), Cytometry 11:418–430; Tsien (1989) in Methods in Cell Biology, Vol. 29 Taylor and Wang (eds.), pp. 127–156). Fluorescently labeled antibodies are particularly useful reporter molecules due to their high degree of specificity for attaching to a single molecular target in a mixture of molecules as complex as a cell or tissue. However, fluorescently labeled antibodies present several limitations.
It is known that luminescent probes can be synthesized within the living cell or can be transported into the cell via several non-mechanical modes including diffusion, facilitated or active transport, signal-sequence-mediated transport, and endocytic or pinocytic uptake. Mechanical bulk loading methods, which are well known in the art, can also be used to load luminescent probes into living cells. (Barber et al. (1996), Neuroscience letters 207:17–20; Bright et al. (1996), Cytometry 24:226–233; McNeil (1989) in Methods in Cell Biology, Vol. 29, Taylor and Wang (eds.) pp. 153–173). These methods include electroporation and other mechanical methods such as scrape-loading, bead-loading, impact loading, syringe-loading, hypertonic and hypotonic loading. Additionally, cells can be genetically engineered to express reporter molecules such as Green Fluorescent Protein, coupled to a protein of interest as previously described (Chalfie and Prasher U.S. Pat. No. 5,491,084; Cubitt et al. (1995), Trends in Biochemical Science, 20:448–455).
Luminescence is the process whereby a molecule is electronically excited and releases light when it returns to a lower energy state. Bioluminescence is the process by which living organisms emit light that is visible to other organisms. In bioluminescence the excited state is created by an enzyme-catalyzed reaction. The color of the emitted light in a bioluminescent reaction is characteristic of the excited molecule, and is independent from its source of excitation and temperature.
Molecular oxygen is known to be essential in some well characterized bioluminescent systems, such as the bioluminescence of luciferase. Luciferases are oxygenases, that act on a substrate, luciferin, in the presence of molecular oxygen and transform the substrate to an excited state. Upon return to a lower energy level, energy is released in the form of light. Ward et al., Chapter 7 in Chemi- and Bio-luminescence, Burr ed. Marcel Dekker, Inc. NY, pp. 321–358; Hastings, J. W. (1995) Cell Physiology: Source Book, N. Sperelakis (ed.), Academic Press, pp. 665–681; Luminescence, Narcosis and Life in the Deep Sea, Johnson Vantage Press, NY, pp. 50–56. Bioluminescent species span many genera and include microscopic organisms, including bacteria, primarily marine bacteria such as Vibrio species, fungi, algae, and dinoflagellates, to marine organisms including arthropods, mollusks, echinoderms, and chordates, and terrestrial organisms including annelids and insects.
Luminescence (bioluminescence, chemiluminescence, and fluorescence) is used for qualitative and quantitative determination of specific substances and processes in biology and medicine. For example, various luciferase genes from various organisms have been cloned and exploited as reporters in numerous assays. On the other hand, treating cells with dyes and fluorescent biomolecules allowing imaging of the cells, and genetic engineering of cells to produce fluorescent proteins as reporter molecules are useful detection methods known by those persons skilled in the art. For instance, treating cells with dyes and fluorescent biomolecules allowing imaging the cells, and genetic engineering of cells to produce fluorescent proteins as reporter molecules are useful detection methods known in the art. Wang et al., Methods in Cell Biology, New York, Alan R. Liss, 29:1–12, 1989. One such fluorescent reporter protein is the green fluorescent protein (GFP) of the jellyfish Aequorea victoria which absorbs blue light with an excitation maximum at 395 nm, with a minor peak at 470 nm, and emits green fluorescence with an emission maximum at 510 nm, with a minor peak near 540 nm and does not require an exogenous factor. However, the absorption and emission spectra for Aequorea GFP present certain limitations. The excitation and emission maxima of the wild type Aequorea GFP are not within the optimal range of wavelengths of standard fluorescence optics.
The green fluorescent proteins (GFP) constitute a class of chromoproteins found among certain bioluminescent coelenterates. These proteins are fluorescent and function as the ultimate bioluminescence emitter in these organisms by accepting energy from enzyme-bound, excited state oxyluciferin. Ward et al., (1982) Biochemistry 21:4535–4540.
Uses of Aequora GFP for the study of gene expression and protein localization are discussed in Chalfie et al., Science 263:802–805, 1994. Some properties of wild-type Aequora GFP are disclosed by Morise et al., Biochemistry 13:2656–2662, 1974, and Ward et al., Photochem. Photobiol. 31:611–615, 1980. An article by Rizzuto et al., Nature 358:325–327, 1992, discusses the use of wild-type Aequora GFP as a tool for visualizing subcellular organelles in cells. Kaether and Gerdes, FEBS Letters 369:267–271, 1995, report the visualization of protein transport along the secretory pathway using wild-type Aequora GFP. The expression of Aequora GFP in plant cells is discussed by Hu and Cheng, FEBS Letters 369:331–334, 1995, while Aequora GFP expression in Drosophila embryos is described by Davis et al., Dev. Biology 170:726–729, 1995.
U.S. Pat. No. 5,491,084 discloses expression of GFP from Aequorea victoria in cells for use as a reporter molecule fused to another protein of interest. PCT/DK96/00052 relates to methods of detecting biologically active substances affecting intracellular processes by utilizing a GFP construct having a protein kinase activation site. GFP proteins are used in various biological systems. For example, PCT/US95/10165 describes a system for isolating cells of interest utilizing the expression of a GFP-like protein. PCT/GB96/00481 describes the expression of GFP in plants. PCT/US95/01425 describes modified GFP protein expressed in transformed organisms to detect mutagenesis. Mutants of GFP have been prepared and used in several biological systems. (Hasselhoff et al., Proc. Natl. Acad. Sci. 94:2122–2127, 1997; Brejc et al., Proc. Natl. Acad Sci. 94:2306–2311, 1997; Cheng et al., Nature Biotech. 14:606–609, 1996; Heim and Tsien, Curr. Biol. 6:178–192, 1996; Ehrig et al., FEBS Letters 367:163–166, 1995). Methods describing assays and compositions for detecting and evaluating the intracellular transduction of an extracellular signal using recombinant cells that express cell surface receptors and contain reporter gene constructs that include transcriptional regulatory elements that are responsive to the activity of cell surface receptors are disclosed in U.S. Pat. No. 5,436,128 and U.S. Pat. No. 5,401,629.
Certain types of cells within an organism may contain components that can be specifically labeled that may not occur in other cell types. For example, epithelial cells often contain polarized membrane components. That is, these cells asymmetrically distribute macromolecules along their plasma membrane. Connective or supporting tissue cells often contain granules in which are trapped molecules specific to that cell type (e.g. heparin, histamine, serotonin, etc.). Skeletal muscle cells contain a sarcoplasmic reticulum, a specialized organelle whose function is to regulate the concentration of calcium ions within the cell cytoplasm. Many nervous tissue cells contain secretory granules and vesicles in which are trapped neurohormones or neurotransmitters. Therefore, fluorescent molecules can be designed to label not only specific components within specific cells, but also specific cells within a population of mixed cell types.
Those skilled in the art will recognize a wide variety of ways to measure fluorescence. For example, some fluorescent reporter molecules exhibit a change in excitation or emission spectra, some exhibit resonance energy transfer where one fluorescent reporter loses fluorescence, while a second gains in fluorescence, some exhibit a loss (quenching) or appearance of fluorescence, while some report rotational movements. (Giuliano et al. (1995), Ann. Rev. of Biophysics and Biomol. Structure 24:405–434; Giuliano et al. (1995), Methods in Neuroscience 27:1–16). The GFPs exhibit absorption at a particular wavelength, and emission at a different wavelength characteristic for each green fluorescent protein which sometimes allows for the pairing of GFP's with two distinct signals being detectable.
In addition to the limitations in detection with standard fluorescence optics presented by the absorption-emission wavelength spectrum of Aequora GFP, another difficulty is the potentially low level of fluorescent signal emitted by GFP transfected into a heterologous cell type. This is the result of low level expression normally associated with the expression of a non-native species protein being expressed by a cell, in this case a jellyfish protein being expressed in higher level organisms such as mammals. This is partly due to different codon usage in the native marine organism sequences that are different from the host or transfected cell's codon usage. In spite of this background art, there remains a very real and substantial need for a fluorescent reporter molecule having a narrower absorption-emission wavelength spectrum and having an optimized expression in a host or transfected cell resulting in fluorescent signals that are easily detected with standard fluorescence optics.
U.S. Pat. No. 5,786,464 (Seed et al.) and U.S. Pat. No. 5,795,737 (Seed et al.) dislcose replacing non-preferred codons with preferred codons to increase expression in mammalian cell lines of other proteins, such as the green fluorescent protein of the jellyfish Aequorea victoria. 
U.S. Pat. No. 5,874,304 (Zolotukhin et al.) discloses a humanized green fluorescent protein gene adapted from the jellyfish Aequorea victoria. U.S. Pat. No. 5,968,750 (Zolotukhin et al.) discloses a method of labeling a mammalian cell comprising expressing a humanized green fluorescent protein gene in the cell wherein the genes have an increased number of GCC or GCT alanine-encoding codons in comparison to the wild type jellyfish gene sequence.
U.S. Pat. No. 6,232,107 (Bryan et al.) discloses isolated and purified nucleic acids encoding green fluorescent proteins from the genus Renilla and Ptilosarcus and the green fluorescent proteins encoded thereby.